Cryptococcus neoformans is a human pathogen that causes life threatening meningoencephalitis in immunocompromised and immunocompetent hosts. The organism is haploid and has a defined sexual cycle, well developed molecular biology research tools, and robust animal models for virulence studies. The basic structure of the genome is well characterized, being about 20MB distributed over 12 to 14 chromosomes. Currently a genome consortium is sequencing the genome of one representative laboratory strain, JEC21. It is anticipated that the JEC21 sequence will be completed within 12 months. [unreadable] [unreadable] There is considerable diversity among C. neoformans isolates. Isolates have been divided into four serotypes which differ by 10% or more at the DNA sequence level. Debate continues over whether these serotypes (A, B, C, D) should be considered different varieties or species. What is important is that these serotypes differ in not only their polysaccharide capsular antigens, but also in physiological properties and in pathogenicity. [unreadable] [unreadable] The most direct and efficient way to address the underlying nature of variation and its relationship to pathogenicity in C. neoformans is a comparative genomics approach taking advantage of the JEC21 sequence, in comparison with the sequence of a serotype A clinical isolate. JEC21 is a laboratory adapted strain derived from a clinical isolate and an environmental isolate by 12 backcrosses and may be attenuated for virulence. We thus propose to determine and annotate the complete sequence of C. neoformans serotype A strain H99. Greater than 90% of all clinical isolates and 99% of all isolates from AIDS patients are of serotype A, and strain H99 is a well characterized clinical isolate. In collaboration with the Vancouver Genome Center, we have produced the preliminary material and results to establish the feasibility of completing the genome in the manner described in this application. We also propose developing the basic resources to exploit this comparative data, namely a careful analysis of the differences between these two strains and a series of microarrays for genotyping of Cryptococcus strains. The resulting data and material from this study will expand our knowledge of the nature of pathogenicity in this important human fungal pathogen. [unreadable] [unreadable]